(1) Role of Energy-dependent Proteases in Protein Quality Control and Cell Regulation. All cells must be capable of degrading aberrant and foreign proteins that would otherwise pollute them. Programmed degradation of regulatory factors also contributes to controlling the cell cycle and to generating peptides for immune presentation. These activities are all carried out by energy-dependent proteases, which generically consist of two parts - a peptidase and a chaperone-like ATPase. [unreadable] (i) For several years, we have studied the Clp proteases of E. coli, considered as a model system. We showed that the peptidase ClpP consists of two apposed heptameric rings and the cognate ATPase - either ClpA or ClpX - is a single hexameric ring. ClpA/X stack axially on one or both faces of ClpP in active complexes. We went on to show that substrate proteins bind to distal sites on the ATPase and are then unfolded and translocated axially into the digestion chamber of ClpP. In FY08, we investigated the N-terminal domains of ClpA that we had previously shown to exhibit high mobility in the region distal to the ATPase ring. Our working hypothesis is that shortening the flexible linker between them will reduce N-domain mobility, rendering them more visible in electron micrographs. The complementary question is: what effect will these truncations have on activity? So far we have analyzed 10- and 15-residue deletion mutants, and find that the effects on N-domain visibility and activity are small, suggesting that the mobile region may be more extensive than previously expected.[unreadable] (ii) The proteosome is the machine responsible for ubiquitin-tagged protein degradation in eukaryotes. The 26S proteasome is composed by the 20s proteolytic chamber and the 19S complex which is involved in substrate binding and translocation into the 20S. The 19S complex contains a hexameric ATPase ring (like ClpA - see above) and numerous regulatory proteins, of which the largest are Rpn1 and Rpn2 (both > 100 kDa). It has been predicted that each has a large domain of PC repeats with alpha-solenoid folds. To evaluate this prediction, we examined the full-length proteins and truncated proteins consistingof the repeat regions by gel filtration, circular dichroism, and negative staining EM. All four proteins are monomeric in solution and highly alpha-helical. The EM data were analyzed by image classification and averaging, showing near-annular molecules, 7 nm in diameter. The full-length and truncated molecules have similar dimensions, indicating that the terminal regions are flexible and thus not visualized. The results supportive of the alpha-toroid model in terms of molecular dimensions and shape, and indicate that the repeats are organized not as symmetrical circular toroids, but in less regular structures.[unreadable] [unreadable] (2a) Intracellular Trafficking: Interaction of Clathrin with Proteins that Regulate its Assembly. Clathrin plays a key role in intracellular trafficking, via its coating of membranous pits and vesicles (CCVs). Assembly of clathrin is promoted by accessory proteins such as auxilin and AP180, and disassembly is effected by the Hsc70 ATPase. In the 1980s, we studied the molecular composition of coated vesicles and the plasticity of the assembly unit, the clathrin triskelion. We returned to this system in FY05, equipped with cryo-EM technology, and compared the structures of coated vesicles with and without binding of the uncoating ATPase, Hsc70. From these observations, we developed a model for uncoating. In FY07, we extended studies initiated during the previous year in which cryo-electron tomography is used to study the structures of individual CCVs isolated from bovine brain. Their polyhedral coats surround cargoes of various shapes and sizes, including vesicles containing neurotransmitters or receptors and viruses. The coated particles reconstructed in the tomograms fall into two sub-populations: 19% have internal vesicles and are true CCVs; the rest 81% lack internal membranes and are "clathrin baskets" (CBs). The CCVs range from 80 to 134 nm in diameter, with vesicles of 30 to 68 nm. The CBs range from 66 to 120 nm. The coats may be described by the symmetry of their polygonal facets. While many small polyhedral forms are possible in theory, many of them are not observed, suggesting that they are energetically disfavored. The common feature of these forbidden small polyhedra is that they have vertices with high curvature. The smallest particle observed is a 28 vertex tetrahedral form, while the smallest CCV has 38 vertices with a 30 nm vesicle. In CCVs, the coat can be coupled to the vesicle via adaptor proteins. In our tomograms we see density between the clathrin N-termini and the membran. The vesicle is always off-center relative to the coat, possibly as a result of budding off during endocytosis. To further investigate the polyhedral forms of coats, we generated a comprehensive set of fullerene polyhedra composed of 12 pentagons and various numbers of hexagons, with from 20 to 60 vertices. These were regularized [unreadable] and used to calculate a variety of energetic measures, such as inter-spar angles, polygon regularity and planarity, curvature [unreadable] and sphericity. This analysis shows that the coat polyhedra are low-energy forms, with more regular polygons and more spherical shapes. Using a simple parameter such as the average inter-spar angular deviation, the probable number of CV [unreadable] polyhedral forms is estimated between 2-5% of all possible 5770 fullerenes with between 20 and 60 vertices.[unreadable] [unreadable] (2b) Intracellular Trafficking: The Retromer Cargo-recognition Complex. The retromer is required for numerous intracellular transactions, such as the sorting of acid hydrolases to lysosomes, transcytosis of the polymeric Ig receptor, Wnt gradient formation, iron transporter recycling, and processing of the amyloid precursor protein. Human retromer consists of two smaller complexes, the cargo-recognition complex and a membrane-targeting complex. We are participating in a study to define the structures of these subcomplexes, their interactions in the fully assembled retromer, and their interactions with targeted membranes. The cargo-recognition complex is a heterotrimer of three proteins, Vps26, Vps29 and Vps35. The crystal structure of a subcomplex of Vps29 and a C-terminal fragment of Vps35 showed that the latter molecule forms a horseshoe-shaped alpha-helical solenoid. From bioinformatic analysis, we infer that the same fold is observed through the rest of Vps35. This prediction is supported by electron microscopy and image processing of the intact Vps26-Vps29-Vps35 complex which is revealed as a somewhat flexible, filamentous structure, 21 nm long. We went on to calculate a 3D reconstruction of the complex at 30 resolution. By docking the known X-ray structures into the 3D map, we were able to build a molecular model for the full cargo-recognition complex. This model displays a 'sequential' arrangement of the three proteins, with vps29 and vps26 on opposite sides and vps35 being the key scaffold for the entire complex, interacting with both vps26 and vps29. This elongated structure presents multiple binding sites for the membrane-targeting complex and receptor cargo.